The hepatic microsomal mixed-function oxidase enzyme system metabolizes a variety of hydrophobic foreign compounds and hormones, and requires substrate, NADPH, and molecular oxygen for activity. The mixed-function oxidase hemoproteins may bind hydrophobic substrates as well as various effector molecules. Although many nitrogen containing substrates and effectors of mixed-function oxidase activity bind to these cytochromes, no systematic study has characterized the reactive site(s) involved in the binding of nitrogenous ligands and their effects on mixed-function oxidase activity. This would be important since man is exposed to many of these nitrogenous compounds in the form of air pollutants, food additives and drugs. I propose to use a reconstituted mixed-function oxidase system containing purified cytochrome P-450 to study the ability of nitrite, organonitrates, and nitrosamines to a) bind cytochrome and alter the absorption spectra; b) compete with known ligands for binding; c) affect the binding of hydroxylation and demethylation suubstrates; d) affect the mixed-function oxidase activity using these substrates; and e) influence the effects of sulfhydryl reagents, sulfhydryl inhibitors, and free radical scavengers on enzyme activity and substrate binding. We are currently concentrating on the effects of indole containing compounds on N-nitrosamine metabolism, in an attempt to determine the factors and mechanisms responsible for N-nitrosamine hepatotoxicity and mutagenicity.